CN103044254A - 2,4-dyhydroxy-6-substituted-phenyl aliphatic ketone derivatives and application thereof - Google Patents
2,4-dyhydroxy-6-substituted-phenyl aliphatic ketone derivatives and application thereof Download PDFInfo
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Abstract
The invention discloses 2,4-dyhydroxy-6-substituted-phenyl aliphatic ketone derivatives and application thereof in preparation of gram negative pathogen resistant inhibitor. Tests confirm that the 2,4-dyhydroxy-6-substituted-phenyl aliphatic ketone derivatives have the effect of obviously inhibiting the type III secretion system of gram negative bacteria; the inhibitors do not inhibit growth of salmonella typhimurium, but is capable of inhibiting invasion of salmonella typhimurium to the host cell, and therefore, the inhibitors can be applied as the type III secretion system inhibitors for gram negative bacteria; and furthermore, the inhibitors can be used as anti-bacterial virulence factor preparations for preparing gram negative pathogen resistant medicines.
Description
Technical field
The present invention relates to class substituted 4-phenyl aliphatic ketone derivative and an application thereof, relate in particular to a class 2,4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative and as the application of bacterial virulence factors inhibitor belongs to organic compound medical applications technical field.
Background technology
At present, along with antibiotic abuse, the resistance of pathogenic bacterium becomes the key subjects of facing mankind gradually, and the continuous appearance of multi-drug resistant bacteria also makes people be faced with the puzzlement that similar microbiotic lacks the age.Therefore, seek new antibiotic and become the extremely urgent problem that needs solution.In the multi-drug resistant bacteria, great majority are conditioned pathogen, and wherein gram negative bacillus accounts for larger proportion, such as intestinal bacteria, shigella, Salmonellas, Pseudomonas aeruginosa etc.Because there is widely host range in gram negative pathogenic bacteria, cause very large healthy and existence to threaten to the mankind, food source property animal and farm crop etc. at present simultaneously, brought huge financial loss.
There is multiple virulence factor in gram negative pathogenic bacteria in pathogenic course, promote it to adhesion or the invasion and attack of host cell, thereby cause further infection.Wherein, three type excretory systems are protein complexes that many gram negative pathogenic bacterias secretion toxic proteins enter host cell, play very important effect aspect pathogenic bacterium pathogenic.There is the conservative type of popularity and structure in three type excretory systems in gram negative pathogenic bacteria, do not exist in host's normal microflora simultaneously, therefore become the antibacterial target of a novelty.Yet, yet there are no report through retrieving 2,4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative as the application of gram negative pathogenic bacteria three type excretory system inhibitor.
Summary of the invention
For the deficiencies in the prior art, the invention provides a class 2,4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative and as the application of gram negative pathogenic bacteria three type excretory system inhibitor.
Of the present invention 2,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative, its chemical structure of general formula is:
Wherein: n=0～8; R=ethyl, methoxyethyl or hydrogen.
In the above-mentioned chemical structure of general formula preferred embodiment be: n=4～6, R=ethyl or methoxyethyl.
Above-mentioned 2,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative most preferably is when n=5, the R=ethyl; When n=6, the R=methoxyethyl.
Above-mentioned 2, the preparation of 4-dihydroxyl-6-substituted 4-phenyl alkanones derivative
With 3; 5-dimethoxyphenylacetic acid 1 is starting raw material; add vitriol oil backflow in the ethanol and obtain 3; 5-dimethoxyphenylacetic acid ethyl ester 2; add again-20 ° of C reactions of boron tribromide and methylene dichloride and obtain 3; 5-dihydroxyphenyl acetic acid ethyl ester 3; then add bromobenzyl and salt of wormwood; reflux in the acetone and obtain benzyl protection product 4; product 4 acidylate under the effect of lipid acid and trifluoroacetic anhydride obtains intermediate 2-acyl group-3; 5-benzyloxy Phenylacetic acid ethylester derivative 5, intermediate 5 is taken off benzyl and is obtained 2-acyl group-3 under the catalysis of palladium carbon, 5-dihydroxyphenyl acetic acid ethyl ester analog derivative C series.
Above-mentioned C series compound refluxes under 2-methyl cellosolve and effect of sulfuric acid and obtains E series derivative.
The decarboxylation of above-mentioned C series compound obtains carboxylated derivative A series.
Above-mentioned preparation feedback route is referring to embodiment 1.
Of the present invention 2, the application of 4-dihydroxyl-6-substituted 4-phenyl alkanones derivative in the anti-gram negative pathogenic bacteria inhibitor of preparation.
Above-mentioned 2, in 4-dihydroxyl-preferred chemical structure of general formula of 6-substituted 4-phenyl alkanones derivative when the R=ethyl, n=4,5,6 or 8; When the R=methoxyethyl, the compound that n=6 explains.
Of the present invention 2,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative can be used as the inhibitor of gram negative pathogenic bacteria virulence factor three type excretory systems and uses.Specifically, as the medicine of antibacterium virulence factor preparation for the preparation of anti-gram negative pathogenic bacteria.
The medicine that treatment gram negative pathogenic bacteria of the present invention infects, wherein: described medicine contains above-mentioned 2,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative and pharmaceutically acceptable auxiliary material.
Food or fodder additives that treatment gram negative pathogenic bacteria of the present invention infects, wherein: described additive contains above-mentioned 2, acceptable auxiliary material in 4-dihydroxyl-6-substituted 4-phenyl alkanones derivative additive preparation.
Acceptable auxiliary material is selected the auxiliary material of this area routine usually in above-mentioned pharmaceutically acceptable auxiliary material or the biotic additives preparation, such as lactose, Microcrystalline Cellulose, methylcellulose gum, Xylo-Mucine, ethyl cellulose, hydroxypropylcellulose, hypromellose (Vltra tears), CAP, HP-55, acetic acid hypromellose succinate, phthalic acid polyvinyl alcohol ester (PVAP), styrene maleic acid multipolymer (StyMA), acrylic resin (enteric solubility I, II, III number), Eudragit L100, Eudragit S 100, cellulose acetate or sodium-chlor and Magnesium Stearate etc.
The present invention proves by restraining effect screening and the Multitest to Salmonellas three type excretory systems secretion toxic protein, find: of the present invention 2,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative can significantly reduce Salmonellas to the invasion and attack of host cell, the three type excretory system inhibitor that can be used as gram negative pathogenic bacteria are used, and further can be used as antibacterium virulence factor preparation for the preparation of the medicine of anti-gram negative pathogenic bacteria.
Of the present invention 2, the effect active and anti-Gram-negative bacteria of the anti-three type excretory systems of 4-dihydroxyl-6-substituted 4-phenyl alkanones derivative is first Application in this area, so for the exploitation broad-spectrum high efficacy and to have a new antibiotic of independent intellectual property right significant.
Description of drawings
Fig. 1: 2,4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative secocurvulin, C5, Csn-B and positive drug INP0403 are to the active testing figure of Salmonella typhimurium invasive ability.
Fig. 2: 2,4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative secocurvulin, C5 and Csn-B are to Salmonella typhimurium growth curve test pattern.
Embodiment
The present invention will be further described below in conjunction with embodiment, and the numbering of wherein said compound is as the criterion to relate to statement in the summary of the invention, and described per-cent number does not specify is mass percent, and institute's raw material that uses does not have being of special instruction commercially available.
Embodiment 1:2, the reaction scheme of 4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative preparation
Agents useful for same and reaction conditions are in the said synthesis route: (i) ethanol, and sulfuric acid refluxes; (ii) boron tribromide, methylene dichloride ,-20 ° of C, 5h; (iii) bromobenzyl, salt of wormwood, acetone refluxes; (iv) lipid acid, trifluoroacetic anhydride; (v) palladium carbon, hydrogen; (vi) 2-methyl cellosolve, sulfuric acid; (vii) sodium hydroxide, ethanol; Hydrochloric acid.
Embodiment 2:C series 2, the preparation of 4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative
The preparation feedback route is referring to embodiment 1.
1g3,5-dimethoxyphenylacetic acid (1) joins in the 30mL dehydrated alcohol, then adds the 1mL vitriol oil, reflux, TLC monitoring reaction process.Add the saturated sodium bicarbonate aqueous solution of 30mL in the 6h afterreaction mixture with neutralisation of sulphuric acid, then use ethyl acetate extraction 3 times, acetic acid ethyl acetate extract with anhydrous sodium sulfate dehydration after, evaporated under reduced pressure.Reactant carries out purifying with ethyl acetate/hexanaphthene gradient elution silicagel column, gets colorless oil (2), yield 95%.MS(ESI):m/z225(M+1)。
0.9g3 5-dimethoxyphenylacetic acid ethyl ester (2) is dissolved in the 20mL anhydrous methylene chloride ,-20 ° of C stir, and then add 18mL BBr
3, stir, TLC monitoring reaction process, after 5 hours, reaction mixture adds the 50mL saturated sodium bicarbonate solution under-20 ° of C, uses ethyl acetate extraction 3 times, then uses anhydrous sodium sulfate dehydration, evaporated under reduced pressure.Reactant carries out separation and purification with ethyl acetate/hexanaphthene gradient elution silicagel column, gets white solid (3), yield 60%.MS(ESI):m/z197(M+1)。
1.4mL being added to, bromobenzyl contains 1g3, in the 50mL acetone soln of 5-dihydroxyphenyl acetic acid ethyl ester (3), and reflux, TLC monitoring reaction process, after 3 hours, reaction mixture is cooled to room temperature, then the pressure reducing and steaming solvent.Reactant adds the entry suspendible, then uses ethyl acetate extraction, behind the anhydrous sodium sulfate dehydration, and evaporated under reduced pressure.Reacting coarse product gets colorless oil 3,5-benzyloxy Phenylacetic acid ethylester (4), yield 92% with ethyl acetate/hexanaphthene gradient elution silica gel column chromatography separates, purifying.3,5-benzyloxy Phenylacetic acid ethylester (4) spectral analysis data:
1H-NMR(600MHz,CDCl
3):δ7.43-7.37(m,8H),7.33(t,J=7.2Hz,2H),6.55(d,J=9.0Hz,2H),5.02(s,4H),4.15(q,J=7.2Hz,2H),3.55(s,2H),1.25(t,J=7.2Hz,3H);
13C-NMR(150MHz,CDCl
3):δ171.4,160.0,136.8,136.1,128.6,127.9,127.5,108.5,100.8,70.1,61.0,41.7,14.41MS(ESI):m/z377[M+H]
+.
1mmol3,5-benzyloxy Phenylacetic acid ethylester (4) lipid acid under the effect of 6mL trifluoroacetic anhydride 40 ° Cs different from 1.2mmol stir, TLC detection reaction process, after 12 hours, under 0 ° of C, neutralize with the 20mL saturated sodium bicarbonate aqueous solution, then use ethyl acetate extraction, extraction liquid with anhydrous sodium sulfate drying after, the evaporated under reduced pressure solvent.Reacting coarse product carries out separation and purification with ethyl acetate/hexanaphthene gradient elution silica gel column chromatography, gets colorless oil 2-carbonyl-3,5-benzyloxy Phenylacetic acid ethylester derivative (5), yield 60～70%.
0.5g different 2-carbonyls-3 after 5-benzyloxy Phenylacetic acid ethylester derivative (5) is used the 30mL dissolve with ethanol, adds 0.5g palladium carbon, stirring at room under the hydrogen effect, TLC monitoring reaction process.React after 12 hours, reaction mixture filters, and then evaporated under reduced pressure separates obtaining white solid C series 2,4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative, yield 89～92% with ethyl acetate/hexanaphthene gradient elution silica gel column chromatography.
Preparation Compound C urvulin: used lipid acid is acetic acid, and intermediate (5) is 2-ethanoyl-3,5-benzyloxy Phenylacetic acid ethylester.
Curvulin
1H-NMR(600MHz,CDCl
3):δ12.14(s,1H),9.57(s,1H),6.35(s,1H),6.26(s,1H),4.16(q,J=7.2Hz2H),3.81(s,2H),2.58(s,3H),1.25(t,J=7.2Hz,3H);
13C-NMR(150MHz,CDCl
3):δ203.2,171.0,164.4,162.2,137.0,116.0,112.7,102.8,61.0,41.5,30.9,14.1;MS(ESI):m/z239[M+H]
+.
Preparation Compound C 1: used lipid acid is n Propanoic acid, and intermediate (5) is 2-positive propionyl-3,5-benzyloxy Phenylacetic acid ethylester.
C1
1H-NMR(600MHz,CDCl
3):δ10.99(s,1H),9.23(s,1H),6.27(dt,J=1.9Hz,1H),6.17(s,1H),4.05(q,J=7.2Hz2H),3.65(s,2H),2.82(q,J=7.2Hz,2H),1.17(t,J=7.2Hz,3H),1.06(t,J=7.2Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.9,171.2,160.9,136.0,111.8,102.6,60.8,36.7,14.2,14.1,8.7;MS(ESI):m/z253[M+H]
+.
Preparation Compound C 2: used lipid acid is butanic acid, and intermediate (5) is 2-positive butyryl radicals-3,5-benzyloxy Phenylacetic acid ethylester.
C2
1H-NMR(600MHz,CDCl
3):δ10.99(s,1H),9.23(s,1H),6.27(d,t,J=1.9Hz,1H),6.17(s,1H),4.05(q,J=7.2Hz,2H),3.65(s,2H),2.82(q,J=7.2Hz,2H),1.17(t,J=7.2Hz,3H),1.06(t,J=7.2Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.93,171.2,160.9,136.0,111.8,102.6,60.8,36.66,14.2,14.2,8.7;MS(ESI):m/z267[M+H]
+.
Preparation Compound C 3: used lipid acid is positive valeric acid, and intermediate (5) is 2-positive pentanoyl-3,5-benzyloxy Phenylacetic acid ethylester.
C3
1H-NMR(600MHz,CDCl
3):δ10.99(s,1H),9.23(s,1H),6.27(d,J=1.9Hz,1H),6.17(s,1H),4.05(q,J=7.2Hz,2H),3.65(s,2H),2.82(q,J=7.2Hz,2H),1.17(t,J=7.2Hz,3H),1.06(t,J=7.2Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.9,171.23，160.9,136.0,111.8,102.6,60.8,36.7,14.2,14.13,8.7;MS(ESI):m/z253[M+H]
+.
Preparation compound S ecocurvularin: used lipid acid is n-caproic acid, and intermediate (5) is 2-positive caproyl-3,5-benzyloxy Phenylacetic acid ethylester.
Secocurvularin
1H-NMR(600MHz,CDCl
3):δ11.97(s,1H),6.32(s,1H),6.26(d,J=2.5Hz,1H),6.24(d,J=2.5Hz,1H),4.21(q,J=7.1Hz，2H),3.82(s,2H),2.82(t,J=7.4Hz,2H),1.71-1.67(m,2H),1.31-1.27(m,7H),0.89(t,J=6.9Hz,2H);
13C-NMR(150MHz,CDCl
3):δ206.7,171.7,164.2,160.3,136.5,116.6,112.7,103.3,61.7,43.4,41.8,31.4,24.6,22.5,14.1,14.0;MS(ESI):m/z295[M+H]
+.
Preparation Compound C 5: used lipid acid is positive enanthic acid, and intermediate (5) is 2-positive oenanthyl-3,5-benzyloxy Phenylacetic acid ethylester.
C5
1H-NMR(600MHz,CDCl
3):δ11.97(s,1H),6.40(s,1H),6.28(s,1H),6.27(s,1H),4.23(q,J=7.0Hz,2H),3.84(s,2H),2.84(t,J=7.0Hz,2H),1.72-1.68(m,2H),1.31-1.29(m,9H),0.89(t,J=6.1Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.76,171.76,164.15,160.29,136.52,116.63,112.72,103.28,61.73,43.44,41.77,31.59,28.93,24.92,22.52,14.16,14.13,14.05;MS(ESI):m/z309[M+H]
+.
Preparation Compound C sn-B: used lipid acid is n-caprylic acid, and intermediate (5) is 2-positive capryloyl-3,5-benzyloxy Phenylacetic acid ethylester,
Csn-B
1H-NMR(600MHz,CDCl
3):δ12.13(s,1H),6.30(d,J=2.4Hz,1H),6.28(d,J=2.4Hz,1H),5.85(s,1H),4.21(q,J=7.1Hz,2H),3.86(s,2H),2.84(t,J=7.4Hz,2H),1.74-1.69(m,2H),1.32-1.6(m,11H),0.90(t,J=6.9Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.7,171.3,164.5,160.1,136.7,116.6,112.5,103.3,61.6,43.4,41.8,31.7,29.2,29.1,25.0,22.6,14.1,14.1;MS(ESI):m/z323[M+H]
+.
Preparation Compound C 8: used lipid acid is n-capric acid, and intermediate (5) is 2-positive decanoyl-3,5-benzyloxy Phenylacetic acid ethylester.
C8
1H-NMR(600MHz,CDCl
3):δ6.27(s,2H),4.21(s,2H),3.81(s,2H),2.84(s,2H),1.69(s,2H),1.29(dd,J=15.6,8.3Hz,15H),0.89(t,J=7.0Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.9,171.9,160.4,136.4,116.8,112.7,103.2,61.7,43.5,41.7,31.9,29.5,29.3,25.0,22.7,14.1;MS(ESI):m/z351[M+H]
+。
Embodiment 3:E series 2, the preparation of 4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative
The preparation feedback route is referring to embodiment 1.
After the C series derivates that 50mg is different is dissolved in the 15mL2-methyl cellosolve, add 2 vitriol oils, stir under 50 ° of C, TLC detection reaction process, 12 – used ethyl acetate extraction after 16 hours, then washed extraction liquid with saturated sodium bicarbonate and sodium chloride solution, ethyl acetate layer with anhydrous sodium sulfate drying after, evaporated under reduced pressure gets reacting coarse product.
With ethyl acetate/hexanaphthene gradient elution silica gel column chromatography separates, purifying, get colorless oil E series derivates, yield 60-80%.
Preparation compd E 0: used C series derivates is Curvulin.
E0
1H-NMR(600MHz,CDCl
3):δ11.64(s,1H),9.51(s,1H),6.26(d,J=2.4Hz,1H),6.15(d,J=2.4Hz,1H),4.15(s,1H),4.21(q,J=7.1Hz,2H),3.73(d,J=12.2Hz,2H),3.48(t,J=4.4Hz,2H),3.26(d,J=12.7Hz,2H),2.46(d,J=12.5Hz,3H);
13C-NMR(150MHz,CDCl
3):δ203.1,171.1,163.5,161.8,136.6,116.7,112.5,102.7,70.2,63.8,43.5,41.0,40.4,40.3,40.1,40.0,39.8,39.7,32.0;MS(ESI):m/z269[M+H]
+.
Preparation compd E 1: used C series derivates is C1.
E1
1H-NMR(600MHz,CDCl
3):δ11.68(s,1H),6.33(d,J=6.5Hz,1H),6.24(d,J=6.5Hz,1H),4.23-4.21(m,2H),3.81(d,J=8.6Hz,2H),3.55(m,3H),3.34(d,J=8.8Hz,2H),2.87(m,2H),1.13(m,3H);
13C-NMR(150MHz,CDCl
3):206.7,171.1,163.3,161.5,136.1,112.5,102.9,70.3,63.9,58.9,41.2,40.3,40.2,40.1,,36.5,8.9;MS(ESI):m/z282[M+H]
+.
Preparation compd E 2: used C series derivates is C2.
E2
1H-NMR(600MHz,CDCl
3):δ11.67(s,1H),7.28(s,1H),7.12(s,1H),6.26(d,J=6.8Hz,1H),6.22(s,1H),4.39-4.28(m,2H),3.85(s,2H),3.66(d,J=3.4Hz,2H),3.42(s,3H),2.83(t,J=7.2Hz,2H),1.78-1.67(m,2H),0.94(dd,J=18.2,10.8Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.7,171.8,163.7,160.5,136.3,116.8,112.6,103.1,70.3,64.2,58.9,45.4,41.4,18.4,13.9;MS(ESI):m/z297[M+H]
+.
Preparation compd E 3: used C series derivates is C3.
E3
1H-NMR(600MHz,CDCl
3):δ12.21(s,1H),6.32(d,J=2.4Hz,1H),6.28(d,J=2.3Hz,1H),6.02(s,1H),4.32-4.27(m,2H),3.91(s,2H),3.40(s,2H),2.86(t,J=7.4Hz,2H),1.74–1.67(m,2H),1.37(dd,J=15.0,7.5Hz,2H),0.94(t,J=7.4Hz,3H);
13C-NMR(150MHz,CDCl
3):δ112.4,103.21,43.1,41.6,27.1,22.3,13.9;MS(ESI):m/z311[M+H]
+.
Preparation compd E 4: used C series derivates is secocurvulin.
E4
1H-NMR(600MHz,CDCl
3):δ11.83(s,1H),6.93(s,1H),6.27(s,1H),6.22(s,1H),4.32(s,2H),3.86(s,2H),3.66(d,J=3.9Hz,2H),3.42(s,3H),2.84(t,J=7.4Hz,2H),1.70(dd,J=14.1,7.0Hz,2H),1.32(t,J=13.4Hz,4H),0.90(t,J=6.7Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.8,171.7,163.9,160.5,136.3,116.7,112.6,103.2,70.3,64.3,58.9,43.4,41.5,31.4,24.6,22.5,14.0;MS(ESI):m/z325[M+H]
+.
Preparation compd E 5: used C series derivates is C5.
E5
1H-NMR(600MHz,CDCl
3):δ11.68(s,1H),7.15(s,1H),6.26(s,1H),6.21(s,1H),4.32(s,2H),3.84(s,2H),3.66(d,J=4.2Hz,2H),3.42(s,3H),2.84(t,J=7.4Hz,2H),1.68(dd,J=14.1,7.0Hz,2H),1.30(d,J=9.6Hz,7H),0.89(t,J=6.6Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.8,171.8,163.7,160.5,136.3,116.8,112.6,103.1,70.3,64.2,58.9,43.5,41.4,31.6,28.9,24.9,22.5,14.1;MS(ESI):m/z339[M+H]
+.
Preparation compd E 6: used C series derivates is Csn-B.
E6
1H-NMR(600MHz,CDCl
3):δ12.10(s,1H),6.29(d,J=2.4Hz,1H),6.24(d,J=2.4Hz,1H),4.34–4.31(m,2H),3.89(s,2H),3.67-3.63(m,2H),3.42(s,2H),2.84(t,J=7.4Hz,2H),1.70(dd,J=14.2,7.2Hz,2H),1.34-1.26(m,7H),0.89(t,J=6.9Hz,3H);
13C-NMR(150MHz,CDCl
3):δ206.8,171.7,171.6,163.8,160.5,136.3,116.7,112.6,103.2,70.3,64.2,58.9,43.5,41.5,31.70,29.2,29.2,25.0,22.6,14.1.MS(ESI):m/z353[M+H]
+。
Embodiment 4:A series 2, the preparation of 4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative
The preparation feedback route is referring to embodiment 1.
50mg C series derivates adds the 1mL sodium hydroxide solution after using the 10mL anhydrous alcohol solution, stirring at room, TLC detection reaction process, after 3～4 hours, reaction solution hydrochloric acid (6M) adjust pH to 2～3 are after reaction mixture ethyl acetate extraction, ethyl acetate layer clean with saturated nacl aqueous solution, use anhydrous sodium sulfate dehydration, then evaporated under reduced pressure.Reaction product is carried out separation and purification with chloroform/methanol gradient elution silica gel column chromatography, gets brown solid A series derivates, yield 80-90%.
Preparation compd A 2: used C series derivates is C2.
A2
1H-NMR(600MHz,DMSO-d
6):δ12.09(s,1H),9.90(s,1H),9.69(s,1H),6.27(d,J=2.0Hz,1H),6.14(d,J=1.9Hz,1H),3.45(d,J=15.6Hz,2H),2.77(t,J=7.3Hz,2H),1.55(m,2H),0.87(t,J=7.4Hz,3H);
13C-NMR(150MHz,DMSO-d
6):δ205.9,172.8,159.6,157.7,136.1,120.7,110.6,101.8,45.9,39.2,17.7,14.3;MS(ESI):m/z239[M+H]
+.
The preparation compound A-13: used C series derivates is C3.
A3
1H-NMR(600MHz,DMSO-d
6):δ12.11(s,1H),9.92(s,1H),9.70(s,1H),6.27(s,1H),6.14(s,1H),3.44(s,2H),2.79(t,J=6.7Hz,2H),1.55(m,2H),1.28(m,4H),0.88(t,J=7.0Hz,3H);
13C-NMR(150MHz,DMSO-d
6):δ206.0,172.8,159.5,157.7,136.0,120.7,110.6,101.8,43.7,39.2,26.5,22.4,14.5;MS(ESI):m/z275[M+H]
+.
Preparation compd A 4: used C series derivates is Secocurvulin.
A4
1H-NMR(600MHz,DMSO-d
6):δ12.11(s,1H),9.88(d,J=41.3Hz,1H),9.67(d,J=41.5Hz,1H),6.27(s,1H),6.14(s,1H),3.44(s,2H),2.78(s,2H),1.53(s,2H),1.26(s,4H),0.88(s,3H);
13C-NMR(150MHz,DMSO-d
6):δ206.0,172.8,159.5,157.7,136.0,120.7,110.6,101.8,43.9,39.2,31.5,24.0,22.5,14.4;MS(ESI):m/z267[M+H]
+.
The preparation compound A-45: used C series derivates is C5.
A5
1H-NMR(600MHz,DMSO-d
6):δ12.10(s,1H),9.91(s,1H),9.70(s,1H),6.27(s,1H),6.14(s,1H),3.43(s,2H),2.78(s,2H),1.52(s,2H),1.27(s,6H),0.87(s,3H);
13C-NMR(150MHz,DMSO-d
6):δ206.0,172.8,159.5,157.7,136.0,120.7,110.6,101.8,43.9,39.2,31.7,28.9,24.3,22.5,14.41;MS(ESI):m/z281[M+H]
+。
Embodiment 5:2, the restraining effect of the anti-three type excretory system toxic proteins secretion of 4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivatives
(referring to document Hudson DL, et.al.Antimicrobe Agents Chemother, 2007.51:2631-2635.)
Test philosophy: three type excretory systems are very important a kind of virulence factors in the Salmonella typhimurium pathogenic course, and it secretes toxic protein to promote the invasion and attack of host cell and the breeding in host cell and diffusion by means of three type excretory systems.Therefore the secretion of blocking toxic protein can suppress Salmonella typhimurium to the invasion of host cell.This experiment is by external evoked method, impel Salmonella typhimurium to pass through three type excretory systems secretion toxic protein to the bacterium liquid of cultivating, then utilize SDS-PAGE and Western Blots to detect, estimate 2,4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative compound to the restraining effect of toxic protein secretion.
Test material:
(1) Salmonella typhimurium: DSMZ of Wuhan University (deposit number CCTCCM91098).
(2) acrylamide-bisacrylamide (40%), APS, TEMED, coomassie brilliant blue R250, the nitrocellulose pvdf membrane is commercially available product.
(3) SipC mouse monoclonal antibody: tgcBioMICS company product;
The goat anti-mouse IgG two of horseradish peroxidase is anti-: Jackson ImmunoResearch company product.
(4) sample preparation: sample is dissolved in before use DMSO and is made into 100mM, and-20 ° of C preserve.
(5) positive control drug: INP0403 (document Hudson DL, et.al.Antimicrobe Agents Chemother, 2007.51:2631-2635 report).
Testing method:
Get the Salmonellas bacteria suspension, be forwarded to LB(0.2%L-ara by 1:100) in, 25 ° of C, the 220rpm shaking culture is spent the night.Then with overnight culture, be forwarded to the fresh LB(0.2%L-ara of 1mL by 1:10) in the substratum, add the medical compounds to be determined that 1 μ l sets concentration, 37 ° of C, 220rpm shaking culture 4h.Get each culture, dilute 10 times, measure OD
600Value.All the other culture 12000g, 4 ° of centrifugal 5min of C.Then gentle aspiration supernatant 800 μ l add 88 μ l100%TCA to final concentration 10%, and vibration is placed 30min on ice several times gently.12000g, 4 ° of centrifugal 5min of C outwell supernatant, every pipe adds the acetone that 400 μ l-20 ° of C preserve, gently vibration several times, 10min on ice, 12000g, 4 ° of centrifugal 5min of C.After volatilizing acetone fully, according to OD
600Be worth, add the sample-loading buffer soluble protein of 1 times of amount of its volume, 95 ° of C heating 5min promote protein denaturation.Get the protein sample for preparing, carrying out 10%SDS-PAGE, then use 0.1%(w/v) coomassie brilliant blue R250 dyes, decolouring, utilizes the gel imaging system imaging, or utilize Western Blots to detect, utilize gel imaging system to carry out the quantitative of imaging and toxic protein SipC.Carry out quantitatively according to the size of SipC spot optical density(OD) among the Western Blots at last, take the secretory volume of blank DMSO group SipC as 100%, calculate the different target compound to the restraining effect of SipC.
Test result sees Table 1:
Table 1. target compound is on the impact of Salmonella typhimurium toxic protein SipC secretion
*The compound test concentrations is 100 μ M.
Can find out that by table 1 result compound S ecocurvulin, C5, Csn-B, C8 and E6 all have obvious restraining effect to the secretion of SipC.
Embodiment 6:2,4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative is to the active testing of Salmonella typhimurium invasion and attack effect
(referring to document Negrea A, et.al.Antimicrobe Agents Chemother, 2007,51:2867-2876.)
Test philosophy:
Salmonella typhimurium is the gram negative pathogenic bacteria of breeding in the born of the same parents, and therefore blocking its invasion and attack to host cell can effectively stop its breeding and diffusion in born of the same parents, thereby reduces pathogenic.This activity test method is take cell in vitro as model, add with 2, the Salmonella typhimurium that the 4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative was processed, then cultivate altogether, calculate invasion and attack by the method for enumeration and enter the number of the bacterium in the host cell, thus the impact of evaluation objective Compounds in Salmonella typhimurium invasive ability.
Test material:
(1) Salmonella typhimurium: DSMZ of Wuhan University (deposit number CCTCCM91098).
(2) HeLa cell: ATCC product.
(3) sample preparation: sample is dissolved in before use DMSO and is made into 100mM, and-20 ° of C preserve.
(5) positive control drug: INP0403 (bibliographical information).
Testing method:
HeLa cell (DMEM, 10%FBS) is inoculated in 24 porocyte culture plates, every hole inoculation 2 * 10
4Individual cell is positioned over 37 ° of C, 5%CO
2Overnight incubation in the incubator.Salmonellas is at LB(0.2%L-ara) 25 ° of C in the substratum, the 220rpm shaken overnight.Then press 1:10 dilution incubated overnight liquid, add target compound to be determined (secocurvulin, C5, Csn-B and positive drug INP0403) 100 μ M, 37 ° of C, 220rpm shaking culture 4h.Get the bacterium liquid of experimental amount, measure OD
600Then value is diluted to setting concentration with DMEM, for subsequent use.The HeLa cell sops up nutrient solution, and with PBS rinse 1 time, then every hole adds 300 μ L DMEM(serum-frees) substratum, behind the preculture 30min, every hole adds 200 μ L dilution bacterium liquid for subsequent use, and (Index of infection is 50, and namely every hole adds 1 * 10
6CFU), 37 ° of C, 5%CO
2Under the condition, cultivate 1h.Then sop up nutrient solution, PBS washes once, and every hole adds the DMEM substratum that 400 μ L contain 100 μ g/ml gentamicins, continues to cultivate 1h, to kill the outer not bacterium of invasion of born of the same parents.Sop up nutrient solution, the HeLa cell cleans three times with PBS, to remove the extracellular bacterium.Every hole adds 200 μ L0.1%TritonX-100, lysing cell.Then the cell pyrolysis liquid gradient dilution is coated diluent respectively in the LB flat board again, and 37 ° of C are inverted overnight incubation, and number goes out single colony number, calculates the colony number that enters the Salmonellas of HeLa cell in every hole.Then calculate invasion and attack rates (%invasion) according to formula, the results are shown in Figure 1.
%invasion=(enters HeLa cell colony number ÷ and adds total colony number in every hole) * 100%
Can find out Compound C 5 by Fig. 1 result, Csn-B and positive control Compound I NP0403 all have extremely significant restraining effect (p≤0.001) to Salmonellas to the invasion of HeLa cell, and compound secocurvulin has significant restraining effect (p≤0.05) to Salmonellas to the invasion of HeLa cell.
Embodiment 7:2,4-dihydroxyl-6-substituted 4-phenyl aliphatic ketone derivative is to the Salmonella typhimurium affects on the growth
Get 25 ° of C overnight culture of Salmonella typhimurium, be forwarded to 1mL LB(0.2%L-ara by 1:10) in, respectively adding target compound C5 to be determined simultaneously, then Csn-B and secocurvulin place 37 ° of C concussions to cultivate.Each sample is set up three repetitions.Every 1h, measure 1 time OD
600, then draw the Salmonella growth curve.
Test result is seen Fig. 2.
As seen from Figure 2, compare with blank DMSO group, target compound is on the almost not impact of growth of Salmonella typhimurium.
Embodiment 8: the medicine that the treatment gram negative pathogenic bacteria infects
The preparation method: Csn-B is mixed with lactose and W-Gum, after water is evenly wetting, sieves, granulation, then dry, after sieve, add Magnesium Stearate, evenly after compressing tablet, every heavy 240mg, Csn-B content is 1mg.
Embodiment 9: the fodder additives that the control gram negative pathogenic bacteria infects
Preparation method: behind 10mg Csn-B and 10g Semen Maydis powder mixing, will remain the complete mixing of Semen Maydis powder and dregs of beans, then with two groups of complete mixings of mixture.Every 1kg chicken feed, Csn-B content is 10mg.
Claims (7)
1. a class 2,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative, its chemical structure of general formula is:
Wherein: n=0～8; R=ethyl, methoxyethyl or hydrogen.
2. as claimed in claim 12,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative is characterized in that: n=4 in the described chemical structure of general formula～6, R=ethyl or methoxyethyl.
3. as claimed in claim 22,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative is characterized in that: described 2,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative is when n=5, the R=ethyl; When n=6, the R=methoxyethyl.
4. claim 1 is described 2, the application of 4-dihydroxyl-6-substituted 4-phenyl alkanones derivative in the anti-gram negative pathogenic bacteria inhibitor of preparation.
5. application as claimed in claim 4 is characterized in that: described 2,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative be in the chemical structure of general formula when the R=ethyl, n=4,5,6 or 8; When the R=methoxyethyl, the compound that n=6 explains.
6. treat the medicine that gram negative pathogenic bacteria infects for one kind, it is characterized in that: described medicine contains claimed in claim 12,4-dihydroxyl-6-substituted 4-phenyl alkanones derivative and pharmaceutically acceptable auxiliary material.
7. treat food or the fodder additives that gram negative pathogenic bacteria infects for one kind, it is characterized in that: described additive contains claimed in claim 12, acceptable auxiliary material in 4-dihydroxyl-6-substituted 4-phenyl alkanones derivative additive preparation.
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| CN115108989A (en) * | 2022-07-04 | 2022-09-27 | 山东大学 | Resveratrol derivative and preparation method and application thereof |
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